Microbial cultures are used extensively in the food, feed and pharmaceutical industry in the manufacturing of fermented products including most dairy products such as cheese, yoghurt and butter, but also in meat, bakery, wine or vegetable products. Furthermore, microbial cultures are also used to produce proteins including enzymes and various kinds of useful compounds. Such microbial cultures are usually referred to as starter cultures and are produced at industrial propagation plants and distributed to the fermentation industry, such as to a dairy plant, where the starter culture is used in their production processes. In particularly cultures of lactic acid bacteria are widely used as starter cultures.
As used herein the term “lactic acid bacterium” (LAB) designates a gram-positive, microaerophilic or anaerobic bacterium which ferments sugars with the production of acids including lactic acid (as the predominantly produced acid), acetic acid and propionic acid. The industrially most useful lactic acid bacteria are found among Lactococcus species (spp.), Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Brevibacterium spp, Enterococcus spp. and Propionibacterium spp. Additionally, lactic acid producing bacteria belonging to the group of the strict anaerobic bacteria, bifidobacteria, i.e. Bifidobacterium spp. which are frequently used as food starter cultures alone or in combination with lactic acid bacteria, are generally included in the group of lactic acid bacteria. Even certain bacteria of the species Staphylococcus (e.g.: S. carnosus, S. equorum, S. sciuri, S. vitulinus and S. xylosus) have been referred to as LAB (Mogensen et al. (2002) Bulletin of the IDF No. 377, 10-19).
The production of LAB starter cultures involves the inoculation of LAB cells in a specific fermentation medium with an appropriate number of the cells to be propagated under appropriate fermentation conditions. In the industrial setting much effort is put into obtaining that the concentration of the propagated cells is high towards the end of the fermentation process. This makes heavy demands on the fermentation conditions and the fermentation medium, which has to support growth of the cells in order to obtain the desired high biomass yields.
The optical density of liquid medium at 600 nm (OD600) is an accurate means of evaluating the density of bacterial cells in a sample of culture. By the term a “high Optical Density-conditions” is referred to fermentations which is characterized by that the concentration of the propagated cells sufficiently high to result in an OD600, which is 10, or more at the end of the fermentation process.
To keep production costs low, industrial fermentations are normally carried out using complex undefined fermentation media. Major components of such media can be yeast extract, cornstarch, whey protein or other milk-based media, which all have complex compositions. For selected fermentation chemically defined media are used which often are made from pure chemicals. Pure chemicals, such as a specific energy or carbon sources, are also often added to complex fermentation media for specific purposes. In either case, the composition of the fermentation medium may be optimal for the viability of the microbial cells, but not optimal for obtaining a high biomass yield of the microorganism.
Most compounds, which are required for cell growth, cost energy for the cell to produce. It often requires that genes encoding the respective biosynthetic enzymes be expressed. The synthesis of these enzymes requires both amino acids and energy. This puts a “protein burden” on the cell, as it must synthesize relatively more enzymes to be able to grow. The precursors required to form the cellular components must furthermore be taken from other pathways, again leading to an additional burden for the cell.
Certain compounds involved in the biosynthesis of nucleic acids have been found to act as so-called cryoprotective agents and reduces the damaging effects on the viability of living cells during freezing and thawing procedures. WO 00/39281 describes the use of inosinate (IMP) and other compounds involved in the biosynthesis of DNA to stabilize the metabolic activity of a liquid starter culture during storage.
It is well-known that LAB have complex growth-factor requirements and that compounds involved in the biosynthesis of DNA and/or RNA stimulate the growth of LAB in chemically defined media. However, several reports show that even though the addition of such compounds result in a shorter lag phase or a higher initial growth rate the addition result in no or only slightly increased yields (Klistrup (2005) FEMS Microbiol Rev. 29, 555-590; Nygaard (1951) J Bact 61, 497-505; Weinman (1964) J Bact 87, 263-269). The addition of compounds involved in the biosynthesis of DNA may even inhibit the yield (Weinman (1964) J Bact 87, 263-269).
As illustrated in examples 3 and 4 the addition of addition of compounds involved in the biosynthesis of DNA also did not increase the biomass yield of fermentations performed high Optical Density-conditions.
Accordingly, there is a need to provide novel approaches to increasing microbial cell biomass yield during fermentation at high Optical Density-conditions.